Abstract

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression.